1. Field of the Invention
The present invention relates to thermocyclers and in particular to thermocyclers for the automated and continuous cycling of fluid between a plurality of temperature zones.
The invention has been developed primarily for use as a thermocycler for nucleic acid amplification and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.
The present invention also relates to a continuous flow system and in particular to a sample port for introducing a volume of a liquid sample into a continuous flow system. However, it will be appreciated that the invention is not limited to this particular field of use.
2. Description of Related Art
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
Systems which require multiple or cyclic chemical reactions to produce a desired product often require careful temperature control, and reproducible and accurate control over the time the reaction is held at temperature. Such reactions include, for example, nucleic acid amplification reactions such as the polymerase chain reaction (PCR) and the ligase chain reaction (LCR).
PCR is a technique involving multiple cycles that results in the geometric amplification of certain polynucleotide sequences each time a cycle is completed. The technique of PCR is well known and is described in many books, including, PCR: A Practical Approach M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification H. A. Erlich, Stockton Press (1989). PCR is also described in many US patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584.
The PCR technique typically involves the step of denaturing a polynucleotide, followed by the step of annealing at least a pair of primer oligonucleotides to the denatured polynucleotide, i.e., hybridizing the primer to the denatured polynucleotide template. After the annealing step, an enzyme with polymerase activity catalyzes synthesis of a new polynucleotide strand that incorporates the primer oligonucleotide and uses the original denatured polynucleotide as a synthesis template. This series of steps (denaturation, primer annealing, and primer extension) constitutes a PCR cycle.
As cycles are repeated, the amount of newly synthesized polynucleotide increases geometrically because the newly synthesized polynucleotides from an earlier cycle can serve as templates for synthesis in subsequent cycles. Primer oligonucleotides are typically selected in pairs that can anneal to opposite strands of a given double-stranded polynucleotide sequence so that the region between the two annealing sites is amplified.
Denaturation of DNA typically takes place at around 90 to 95° C., annealing a primer to the denatured DNA is typically performed at around 40 to 60° C., and the step of extending the annealed primers with a polymerase is typically performed at around 70 to 75° C. Therefore, during a PCR cycle the temperature of the reaction mixture must be varied, and varied many times during a multicycle PCR experiment.
A number of thermal “cyclers” used for DNA amplification and sequencing are disclosed in the prior art in which one or more temperature controlled elements or “blocks” hold the reaction mixture, and wherein the temperature of the block is varied over time. These devices suffer the drawback that they are slow in cycling the reaction mixtures and temperature control is less than ideal. In an effort to overcome the need to cyclically raise and lower the temperature of the heating blocks, others have devised apparatus known in the art as a thermocycler. In this apparatus, multiple temperature controlled blocks are kept at different desired temperatures and a robotic arm is utilized to move reaction mixtures from block to block. Typical thermocycler systems are disclosed in U.S. Pat. Nos. 5,443,791; 5,656,493 and 6,656,724. However, as will be appreciated, these systems suffer from their own set of drawbacks. For example, they have a relatively limited throughput, they are physically large, prone to break down, expensive and require constant routine maintenance.
Difficulties encountered with these aforementioned devices were in part addressed by the invention disclosed in U.S. Pat. No. 5,270,183. In essence, that invention was directed to the reactants travelling through a continuous tube which was subjected to varying temperatures by coiling the tube around substantially drum shaped bodies held at varying temperatures. In order to prevent cross contamination between samples, the reaction mixture was injected into a stream of carrier fluid which separated individual reaction mixtures and passed through two or three separate heating zones. The carrier fluid and reaction mixture are immiscible such that each sample is cleanly separated from the preceding and following sample by segments of carrier fluid. This arrangement allows sequential processing of a number of samples. However, this device has drawbacks, for example since the heating zones are spatially separated it is not convenient to conduct real-time monitoring of the course of a reaction. In addition, having heating zones separated from each other physically tends to be cumbersome.
An advance over the invention disclosed in U.S. Pat. No. 5,270,183 is described in WO 03/016558 in which a single drum shaped body is longitudinally divided to provide at least two segments which are able to be heated to different temperatures such that the body may have peripheral surfaces of different temperatures. As the reactants travel through the continuous tube coiled around the body the reactants are subjected to alternating temperatures. However, the problem with such a thermocycler is that it is difficult to obtain a sufficiently rapid readout of data using the linear tracking device, which scans the periphery of the drum.
With respect to continuous flow systems and apparatus generally, including thermocyclers described above, they are typically operated under positive pressure and require pumps for pumping a carrier fluid through a continuous tube/conduit such as a reaction tube. Typically, these pumps are high or very high pressure pumps. Accordingly, these prior art continuous flow apparatus require specialised high-pressure injection ports for delivering a liquid sample into the stream of carrier fluid being pumped under high pressure through the tube. These high-pressure injection ports suffer a variety of drawbacks. However, the major drawback is their propensity for cross contamination between samples such as for example contamination of samples during loading, largely due to the septum-needle arrangement for injecting the sample, or between samples as they travel down the tubing.
Thus there still remains a need for improved continuous flow systems, including thermocycler devices as described herein, as well as sample handling and delivery means.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the abovementioned prior art, or to provide a useful alternative.